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Pharmacology & Pharmacy, 2015, 6, 25-33 Published Online
January 2015 in SciRes. http://www.scirp.org/journal/pp
http://dx.doi.org/10.4236/pp.2015.61004
How to cite this paper: Reina, S., Pisoni, C., Eimon, A.,
Carrizo, C., Arana, R. and Borda, E. (2015) Anti-M3 Muscarinic
Ace-tylcholine Receptor Antibodies in Systemic Lupus Erythematosus.
Pharmacology & Pharmacy, 6, 25-33.
http://dx.doi.org/10.4236/pp.2015.61004
Anti-M3 Muscarinic Acetylcholine Receptor Antibodies in Systemic
Lupus Erythematosus Silvia Reina1,2, Cecilia Pisoni3, Alicia
Eimon3, Carolina Carrizo3, Roberto Arana3, Enri Borda1,2*
1Pharmacology Unit, School of Dentistry, University of Buenos
Aires, Buenos Aires, Argentina 2National Research Council of
Argentina (CONICET), Buenos Aires, Argentina 3Section of
Rheumatology and Immunology, Department of Internal Medicine,
CEMIC, Buenos Aires, Argentina Email: *[email protected]
Received 19 December 2014; accepted 16 January 2015; published 20
January 2015
Copyright © 2015 by authors and Scientific Research Publishing
Inc. This work is licensed under the Creative Commons Attribution
International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Abstract Background: Evidences have shown that anti-M3
muscarinic acetylcholine receptor IgG (anti-M3 mAChR IgG) are
clinically useful autoantibody that exert a cholinergic
pharmacologic effect bind-ing and interacting with M3 mAChR at the
level of exocrine gland (salivary and ocular). Aims: The aim of
this study was to determine the associations between serum level of
anti-M3 mAChR IgG in patients with systemic lupus erythematosus
(SLE) and other autoantibodies, serum prostaglandin E2 (PGE2), and
clinical manifestations. Methods: Serum autoantibodies against M3
mAChR synthetic peptide were measured by enzyme-linked immuno
absorbent assay (ELISA) using, as an antigen, a 25-mer peptide
K-R-T-V-P-D-N-Q-C-F-I-Q-F-L-S-N-P-A-V-T-F-G-T-A-I corresponding to
the amino ac-id sequence of the second extracellular loop of the
human M3 mAChR. Serum levels of antinuclear antibodies (ANA),
anti-Smith (Sm) antibodies, anti-phospholipid (APL) antibodies, and
PGE2 were determined by ELISA in patients with SLE. Results: We
found significantly enhanced titers of an-ti-M3 mAChR IgG in sera
from SLE patients compared with healthy individuals (control). In
addi-tion, serum levels of PGE2 were significantly higher in SLE
patients than in control patients and were significantly higher in
active than in non-active SLE. No correlation was found with other
au-toantibodies present in SLE. By contrast, a positive correlation
was found between anti-M3 mAChR IgG and PGE2 serum levels in SLE.
Conclusions: As anti-M3 mAChR antibodies present in the sera of SLE
patients may be another factor in the pathogenesis of this disease,
and the increment of PGE2 in the sera of SLE has a modulatory
action on the inflammatory process, suggesting that the pres-ence
of these autoantibodies against M3 mAChR may contribute to
sustained immune deregulation and the strong inflammatory component
observed in SLE.
*Corresponding author.
http://www.scirp.org/journal/pphttp://dx.doi.org/10.4236/pp.2015.61004http://dx.doi.org/10.4236/pp.2015.61004http://www.scirp.orgmailto:[email protected]://creativecommons.org/licenses/by/4.0/
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S. Reina et al.
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Keywords Anti-M3 mAChR Antibodies, Systemic Lupus Erythematosus,
Prostaglandin E2
1. Introduction The onset and development of autoimmune disease
(AID) are the consequence of interactions between genetic and
environmental factors, which result in dysregulation of the immune
system, and are characterized by the presence of autoantibodies and
autoreactive T cells. Under these circumstances, immune system
antimicrobial defenses react against normal components of the body
and result in organ-specific or systemic immunopatholo-gy.
Autoantibodies may also appear in the blood of healthy individuals
or in some special situations, such as in-fection and the
pre-clinical phase of infectious diseases [1]-[3].
Systemic lupus erythematosus (SLE) is an autoimmune disease
characterized by the production of multiple autoantibodies [4]-[8],
with an inflammatory/necrotic phenomenon in different tissues [9]
[10]. This condition is associated with hyperactivity of B cells,
and different immuno-regulatory abnormalities [11] [12]. In
addition, T cells from SLE patients exhibit defects in early
activation events as well as an impaired proliferative response to
mitogenic lectins [12] [13]. Furthermore, anti-nuclear (ANA),
anti-Smith (anti-Sm), anti-phospholipid (APL), and other
autoantibodies are detected in patients with SLE [4] [14] [15]. One
early study, in which 23 asympto-matic pregnant women with positive
anti-Ro or anti-La titers were followed for many years, reported
that four subjects developed SLE, which suggested that anti-Ro or
anti-La antibodies preceded the development of SLE [16]. In another
study, Aho et al. reported that SLE patients were positive for ANA
before the onset of SLE and the percentage was much higher than
that of controls which was a much higher rate than controls [17].
Moreover, the results from the United States Department of Defense
Serum Repository showed that the presence of ANA, anti-Ro, anti-La,
anti-double strain deoxyribonucleic acid (anti-ds DNA), anti-Sm,
APL, anti-ribonucleo protein (anti-nRNP) antibodies and rheumatoid
factor (RF) preceded the onset of SLE [18]-[20]. Among SLE
patients, 88% were positive for at least one of these
autoantibodies, which was a much higher percentage than healthy
controls, and the prevalence of these autoantibodies increased
after diagnosis [21] [22]. Anti-Ro, anti-La, and APL antibodies
were the earliest detectable autoantibodies during the pre-clinical
phase of rheumatoid arthritis (RA) [18]-[22]. In addition, the
presence of these autoantibodies was associated with incipient
severe SLE. For example, patients who were positive for anti-ds DNA
antibodies often developed renal disease [22] [23], pa-tients who
were positive for IgG RF were more likely to develop arthritis
[18], and positive APL was associated with malar rash and
photosensitivity [19]. In addition, regular patterns exist among
these autoantibodies. The majority of La-positive pre-clinical SLE
patients were also Ro-positive, and a significant overlap was
observed between patients positive for anti-ds DNA antibody and
those positive for anti-chromatin antibody [20].
A recently published nested case-control study showed that 66%
of Sjögren Syndrome (SS) patients were positive for ANA, RF, and
anti-La or anti-Ro antibodies approximately 5 years before the
onset of SS. A maxi-mum of 18 years elapsed between positivity for
these antibodies and the onset of SS in these subjects [23]. The
seropositive rate for these antibodies and the anti-M3 muscarinic
acetylcholine receptor (mAChR) autoantibo-dies increased as the
onset of SS neared [24]-[26]. In conclusion, the evidence suggests
that these autoantibodies are predictors of SS. Moreover, the
presence of these autoantibodies in the serum of patients with SLE
is another topic of discussion in SLE pathogenesis.
The autoimmune nature of lupus and its predominant inflammatory
component was accompanied by the ex-pression of cyclo-oxygenase-2
(COX-2) in the inflammatory areas, with the subsequent release of
arachidonic acid via membrane-bound phospholipase A2. The
biosynthesis of arachidonic acid by COX-2 led to an en-hancement of
prostanoid production of PGE2 serie, which conduct to the
dysregulation in the production of proinflammatory cytokines (IL-6,
IL-10, and nitric oxide) [27] [28]. Therefore, we focused on the
family of prostaglandins (PG), which are the result of the
oxidative modification of arachidonic acid and its cascade products
through COX-2 expression and activation in patients with SLE.
2. Aim The aim of our preliminary study was to investigate the
inflammatory status in SLE patients compared to active and
non-active groups by assessing the generation of PGE2 and its
association with the presence or absence of
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S. Reina et al.
27
anti M3 mAChR autoantibodies and SLE disease activity index
(SLEDAI).
3. Methodology 3.1. Patients Blood samples from 30 patients with
SLE, according to the classification criteria of the American
College of Rheumatology (ACR) [29] were obtained. A total of 26
women subjects and four men subjects with a mean age of 41.4 ± 11.9
years were included in the study. Also, blood samples of 30 healthy
women subjects with a mean age of 39.6 ± 10.2 years were used as
controls. Fifteen patients had non-active disease (
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S. Reina et al.
28
peptide antibodies were then eluted with 3 M potassium
thiocyanate (KSCN) and 1 M sodium chloride (NaCl), followed by
immediate extensive dialysis against PBS. The IgG concentrations of
non-anti-peptide antibodies and specific anti-muscarinic receptor
peptide IgG were determined by a radial immunodiffusion assay, and
their immunological reactivity against muscarinic receptor peptides
was evaluated by ELISA. The concentration of the affinity-purified
anti-M3 peptide IgG (1 × 10–7 M) that maximally increased optical
density (OD: 2.4 ± 0.2) corresponded to a total IgG concentration
of 1 × 10–6 M (OD: 2.2 ± 0.2). The normal IgG fraction purified by
af-finity column chromatography gave a negative result (OD: 0.24 ±
0.03).
3.4. ELISA Assay Fifty microlitres of M3 mAChR peptide solution
in 0.1 M sodium carbonate (Na2CO3) buffer (pH = 9.6) was used to
coat microtiter plates (Corning Costar, Tewksbury, MA, USA) at 4˚C
overnight as decribed [25]. After blocking the wells, varying
concentrations of purified IgG from patients with SLE and healthy
individuals were allowed to react with the antigens for 2 h at
37˚C. The wells were then thoroughly washed with Tween® 20 in PBS.
Goat anti-human IgG avidin-alkaline phosphatase (50 μl) was added
and incubated for 1 h at 37˚C. After several washing steps,
p-nitrophenyl phosphate (1 mg∙mL−1) was added as the substrate; the
reaction was termi-nated at 30 min. OD values were measured using
an ELISA reader (Uniskan Laboratory System, Helsinki, Fin-land). As
negative controls, non-antigen paired wells and wells with no
primary antiserum were also tested.
3.5. PGE2 Procedure Serum PGE2 was measured by ELISA according
to the manufacturer’s protocol (PGE2 Biotrack Enzyme Im-mune Assay
System; Amersham Bioscience, Piscataway, NJ, USA). The OD cutoff
value for PGE2 was 4.4 ± 0.33 ng/ml. All serum samples were frozen
promptly after collection and kept at −80˚C until used for PGE2
de-termination. The PGE2 results are expressed as ng/mL.
3.6. Statistical Analysis Statistical analysis was performed
using GraphPad Prism (GraphPad, San Diego, CA, USA). Statistical
signi-ficance was determined by the two-tailed t test for
independent populations. Analysis of variance (ANOVA) and Dunn’s
and Kruskal-Wallis tests were employed for multiple comparisons.
Pearson’s analysis was applied to establish correlation.
Differences between means were considered significant at P ≤
0.05.
3.7. Ethical Approval of the Study Protocol The study was
approved by the Ethics Committee of the School of Dentistry at
Buenos Aires University (Bu-enos Aires, Argentina). The studies
were conducted according to the tenets of the Declaration of
Helsinki. All participants provided written informed consent to
participate in the study.
4. Results ELISA assays were performed to determine whether a
correlation exists between serum IgG against M3 mAChR synthetic
peptide (Figure 1(a)) as well as serum PGE2 levels (Figure 1(b)) in
SLE patients compared with nor-mal individuals. Figure 1(a) shows
the optical density (OD) values for each studied serum from SLE
patients and normal subjects. Also, Figure 1(b) shows serum PGE2
levels in SLE patients and normal subjects. The cut-off values
obtained with SLE patient sera were always greater than two
standard deviations (SD) from those from normal individuals. In
Figure 1(c), a positive correlation is shown between serum PGE2
levels and anti-M3 synthetic peptide titers (see table insert) of
the individual sera from SLE patients.
Additionally, Table 2 shows the comparison and a statistical
analysis of different autoantibodies from sera of SLE patients,
showing that anti-M3 synthetic peptide IgG was significantly
different from anti-Sm antibodies and APL antibodies. No
significant values were obtained when we compared anti-M3 synthetic
peptide IgG with ANA.
The possible association between SLE disease activity, according
to the SLEDAI and levels of anti-M3 syn-thetic peptide IgG and
serum PGE2 levels (expressed as optical density values or ng/ml,
respectively) was tested. Accordingly, when SLE patients were
grouped on the basis of the disease status (active or non-active),
no sig-
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S. Reina et al.
29
Figure 1. Scattergram showing immunoreactivity of circulating
IgG antibodies against M3 mAChR synthetic peptide (a) and serum
PGE2 (b). The individual optical density (OD) values for each serum
sample (1:30 dilution) from 30 SLE patients and 30 normal
individuals. Dot-ted/dashed line: cutoff values of OD 0.24 and 4.40
for anti-M3 mAChR synthetic peptide IgG and serum PGE2,
respectively. Solid lines, median OD values. P < 0.0001 between
groups, and (c) correlation between the anti-M3 synthetic peptide
IgG and serum PGE2 levels.
nificant differences were found for the anti-M3 synthetic
peptide IgG (Figure 2(a)). On the contrary, a signifi-cant
association between active or non-active status of SLE patients was
observed when we analyzed serum PGE2 levels (Figure 2(b)).
Finally, when we examined the possible association among the
three parameters tested (SLEDAI, anti-M3 synthetic peptide IgG
levels, and serum PGE2 levels) in this study, a highly significant
association was found
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S. Reina et al.
30
Table 2. Comparison between anti-M3 synthetic peptide IgG and
different antibodies in SLE patients.
Variable Dunn’s multiple comparison test Number of patients
Anti-M3 synthetic peptide IgG versus ANA ns 30
Anti-M3 synthetic peptide IgG versus anti-Sm Yes (P < 0.05)
30
Anti-M3 synthetic peptide IgG versus APL Yes (P < 0.05)
30
Results were analyzed by one-way ANOVA followed by
Kruskal-Wallis tests (P < 0.0001).
Figure 2. Scattergram showing immunoreactivity of circulating
IgG antibodies against M3 mAChR synthetic peptide (a) and serum
PGE2 (b) in active and non-active forms of SLE disease. Values are
the individual OD values for each serum sample from 30 SLE patients
and 30 healthy volunteers. Dotted/dashed line: cutoff values of OD
0.24 and 4.40 for anti-M3 mAChR synthetic peptide IgG and serum
PGE2, respectively. Solid lines, median OD values. P < 0.0001
between groups.
only between the levels of PGE2 and the active and non-active
SLE disease status (SLEDAI) (Table 3).
5. Discussion Systemic lupus erythematosus (SLE) is a
challenging disease to assess and manage. Much progress in our
un-
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31
Table 3. Association between disease activity (SLEDAI) and
levels of autoantibodies and serum PGE2.
Conditions Active SLE Non-active SLE P values
SLEDAI 6.51 ± 0.90 0.53 ± 0.23
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32
in patients with SLE, as well as significantly higher levels of
serum PGE2. We consider that PGE2 and these cholinergic
autoantibodies may contribute, in part, to the pathogenesis of the
autoreactive and inflammatory phenomenon described here and
observed in SLE patients.
Acknowledgements This study was supported by grants from the
University of Buenos Aires (UBACyT 2011-2014) and CONICET (PIP
2014).
Conflict of Interest The authors declare that they have no
vested interest that could be constructed to have inappropriately
influ-enced this study.
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Anti-M3 Muscarinic Acetylcholine Receptor Antibodies in Systemic
Lupus ErythematosusAbstractKeywords1. Introduction2. Aim3.
Methodology3.1. Patients3.2. Autoantibody Detection3.3. M3 mAChR
Autoantibodies3.4. ELISA Assay3.5. PGE2 Procedure3.6. Statistical
Analysis3.7. Ethical Approval of the Study Protocol
4. Results5. Discussion6. ConclusionAcknowledgementsConflict of
InterestReferences
